Neo lite luciferase assay system

Ad26 and Ad35 NAb titers in serum were assessed using a luciferase-based VNA based on the method described in ref. ^{61 (link)} A549 human lung carcinoma cells (catalog number ATCC CCL-185, obtained from the American Type Culture Collection, Manassas, VA) were plated at a density of 1 × 10⁴ cells/well in 96-well black-and-white isoplates (Wallac, Turku, Finland). E1/E3-deleted Ad26- or Ad35-luciferase reporter constructs were then added at a multiplicity of infection (MOI) of 500, together with twofold serial dilutions of individual heat-inactivated cynomolgus macaque serum samples starting at a 1:64 dilution for Ad26, or a 1:32 dilution for Ad35 in 200 μl reaction volumes. After incubation for 24 h at 37 °C and 10% CO₂, luciferase activity in cryolysed cells was measured using the Neo-Lite Luciferase Assay System (PerkinElmer, Groningen, Netherlands) on a BioTek Synergy Neo luminescence counter (BioTek). Ninety percent neutralization titers (IC90) were defined as the maximum serum dilution that neutralized 90% of luciferase activity. Each serum sample was analyzed in duplicate.

Ad26 Nab titers in serum were assessed using a luciferase-based virus neutralization assay (VNA)^{50 (link)}. Briefly, heat-inactivated Cynomolgus Macaque serum samples was 2-fold serial diluted starting at a 1:32, 1:64 or 1:128 dilution for Ad26 (depending on the study, see figure legends for the exact start dilution). E1/E3 deleted Ad26-luciferase reported constructs were combined with serial diluted serum into a Tissue Culture treated Black and White Isoplate-96 (Perkin Elmer, Nederland B.V) at 500 to 1000 vp/cell. Plates were incubated for 30 minutes at RT, before A549 human lung carcinoma cells (ATCC^® CCL-185™, American Type Culture Collection, Manassas, VA, USA) were added at a density of 1×10⁴ cells/well. After incubation for 20 to 24 hours at 37 °C and 10% CO₂, luciferase activity was measured using the Neo-Lite Luciferase Assay System (Perkin Elmer, Waltham, MA, USA) and a BioTek Synergy Neo luminescence counter (BioTek/Agilent, Santa Clara, CA, USA) or EnVision multimode plate reader (Perkin Elmer). A 90% neutralization titer (IC₉₀) was defined as the maximum serum dilution that neutralized 90% of luciferase activity. Each serum sample was analyzed in duplicate.